Method for the production of conjugates and uses thereof for the prevention and treatment of allergic reactions and autoimmune diseases

ABSTRACT

The present invention relates to a method for the preparation of a conjugate comprising a first and a second polypeptide, said method comprising the steps of (a) incubating said first polypeptide in the presence of a heterobifunctional crosslinker comprising an N-hydroxylsuccinimide ester group and a maleimide group linked via a polyethylene oxide spacer; (b) removing excess heterobifunctional crosslinker; and (c) incubating the reaction product of step (b) with said second polypeptide, wherein said second polypeptide comprises at least one sulfhydryl group. 
     Furthermore, the present invention relates to a conjugate obtainable by the method of the present invention. Also described is a pharmaceutical composition comprising the conjugate of the present invention and, optionally, a pharmaceutically acceptable carrier and/or diluent, and the use of the conjugate for the preparation of a pharmaceutical composition for preventing and/or treating an allergic disease or an autoimmune disease.

The present invention relates to a method for the preparation of aconjugate comprising a first and a second polypeptide, said methodcomprising the steps of (a) incubating said first polypeptide in thepresence of a heterobifunctional crosslinker comprising anN-hydroxylsuccinimide ester group and a maleimide group linked via apolyethylene oxide spacer; (b) removing excess heterobifunctionalcrosslinker; and (c) incubating the reaction product of step (b) withsaid second polypeptide, wherein said second polypeptide comprises atleast one sulfhydryl group. Furthermore, the present invention relatesto a conjugate obtainable by the method of the present invention. Alsodescribed is a pharmaceutical composition comprising the conjugate ofthe present invention and, optionally, a pharmaceutically acceptablecarrier and/or diluent, and the use of the conjugate for the preparationof a pharmaceutical composition for preventing and/or treating anallergic disease or an autoimmune disease.

Immunologic tolerance may be defined as a state of antigen-specificunresponsiveness induced by preexposure to an antigen. If the antigen isan allergen, the immune response is defined as allergy, an adversereaction with an immunologic basis mediated by IgE immunoglobulin(Sampson (1986), J. Allergy Clin. Immunol. 78:212-219). The immunesystem may also be a cause of disease or other undesirable consequences,when the principle of self/non-self recognition breaks down and thebody's own components are recognized as non-self (autoantigens) in whichcase autoimmune diseases can ensue.

Interest in immunologic tolerance, discovered by Medawar almost half acentury ago (Billingham et al. (1953), Nature 172:603-606), hasincreased for two main reasons: (1) Several of its mechanisms, such asclonal deletion (Kappler et al. (1987), Cell 49:273-280), anergy(Jenkins and Schwartz (1987), J. Exp. Med. 165:302-319), and regulatoryT cells (Gershon and Kondo (1971), Immunology 21:903-914) have beenuncovered. (2) Both systemic and oral tolerance (Cremer et al. (1983),J. Immunol. 131:2995-3000; Weiner et al. (1994), Ann. Rev. Immunol.12:809-837) can be induced to, it is hoped, prevent either autoimmune orallergic diseases. For example, several strategies have been used intrying to prevent allergy, including administration of modified allergen(Lee and Sehon (1977), Nature 267:618-649), allergen linked tononimmunogenic carriers (Katz et al. (1971), J. Exp. Med. 134:201-203),single peptides (Muckerheide et al. (1977), J. Immunol. 119:1340-1345),or an allergen-antibody complex (Machiels et al. (1990), J. Clin.Invest. 85:1024-1035).

It is known that antigen presentation can influence the type of immuneresponse. Not only haptens (Borel (1989), in “Concepts inImmunopathology”, Cruse and Lewis (Eds.), 7:145-161, Karger, Basel;Sehon (1982), Prog. Allergy 32:161-202) but also proteins covalentlylinked to a carrier molecule naturally tolerated by the host, such asisologous immunoglobulin, can induce unresponsiveness to these proteins(Filion et al. (1980), Cell Immunol. 54:115-128; Borel and Borel (1990),J. Immunol. Methods 126:159-168).

However, although the above strategies proved to be partiallysuccessful, there is still a need for allergen and/or auto-antigencomprising conjugates with improved therapeutic properties.

Thus, the technical problem underlying the present invention was toprovide a method for the production of such allergen and/or auto-antigencomprising conjugates.

The solution to this technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the present invention relates to a method for thepreparation of a conjugate comprising a first and a second polypeptide,said method comprising the steps of:

(a) incubating said first polypeptide in the presence of aheterobifunctional crosslinker comprising an N-hydroxylsuccinimide estergroup and a maleimide group linked via a polyethylene oxide spacer;

(b) removing excess heterobifunctional crosslinker; and

(c) incubating the reaction product of step (b) with said secondpolypeptide, wherein said second polypeptide comprises at least onesulfhydryl group.

It is envisaged in accordance with the present invention that thepolyethylene oxide spacer may consist of from 1 to 10 monomer units.Preferably, the spacer consists of from 2 to 5 monomer units.

Unexpectedly, it has been found in accordance with the present inventionthat due to the crosslinker used conjugates prepared by the method ofthe present invention show superior features as compared to conjugatesprepared by prior art methods. For example, conjugates of ragweedderived antigen (Amba-l) and mouse IgG were prepared by the method ofthe present invention and a method using as crosslinkerN-(γ-maleimidobutyroxy)sulfosuccimimide ester (sulfo-GMBS) a preferredprior art crosslinker (see Example 1, infra). The effects of bothconjugates in the treatment of an allergic reaction to ragweed derivedantigen were investigated. Surprisingly, it was found that the conjugateof the present invention leads to a significant reduction in airwayhyperresponsiveness whereas the prior art conjugate has almost no effectwhen compared to sensitized animals (see Example 5 and FIG. 3).Furthermore, the conjugate of the present invention virtually eliminatedeosinophils in the bronchoalveolar lavage fluid (BALF). This phenomenonwas accompanied by a total suppression of specific anti-Amba-l IgE. Incontrast, the prior art conjugate only moderately reduced eosinophils,and specific anti-Amba-l IgEs were only partially suppressed (seeExample 5 and FIG. 4). These results clearly demonstrate that due to thecrosslinker used the method of the present invention allows the personskilled in the art to produce conjugates with advantageous immunologicalproperties which render these conjugates for instance suitable for thedownregulation of the inflammatory and immunologic reactions of anallergic response.

In a preferred embodiment of the present invention, said at least onesulfhydryl group of said second polypeptide is introduced by:

(i) incubating said second polypeptide in the presence ofN-succinimidyl-S-acetylthioacetate (SATA);

(ii) removing excess SATA;

(iii) incubating the reaction product of step (ii) in the presence ofhydroxylamine; and

(iv) removing excess hydroxylamine and acetylated hydroxylamine.

This embodiment is of particular importance in cases where said secondpolypeptide does not comprise at least one endogenous sulfhydryl groupthat is, for example, provided by a cysteine residue in the amino acidsequence, and which is suitable for crosslinking said second polypeptidewith said first polypeptide via a disulfide bond. Sulfhydryl groups thatare not suitable for crosslinking may be, for example, sulfhydryl groupsthat are not readily accessible for the sulfhydryl group of said firstpolypeptide due to the three dimensional conformation of said secondpolypeptide, or that are not available due to inter- or intramoleculardisulfide bonds. Whether said second polypeptide comprises one or moresulfhydryl groups that allow effective crosslinking to occur or whethersulfhydryl groups have to be introduced into said second polypeptide canbe determined by the person skilled in the art without further ado. Forexample, two polypeptides may be crosslinked by the method of thepresent invention and the quality and quantity of the obtained conjugatemay be analyzed by SDS polyacrylamide gel electrophoresis undernon-reducing conditions and immunoblotting with an antibody specific forone of the two crosslinked polypeptides. Preferably, the identity of theconjugate is verified with a second antibody specific for the second ofthe two polypeptides. For a detailed description of the above outlinedexperiment, reference is made to Example 1. If the results show that thequality and quantity of the conjugate is not satisfactory as compared toa reference conjugate like, for example, the IgG/Amba-l conjugateproduced in Example 1, sulfhydryl groups may be introduced as describedabove.

In another preferred embodiment, said heterobifunctional crosslinkercomprising an N-hydroxylsuccinimide ester group and a maleimide grouplinked via a polyethylene oxide spacer has the formula:

In a further preferred embodiment of the method of the presentinvention, the heterobifunctional crosslinker is used in step (a) in a5- to 50-fold higher molar concentration than said first polypeptide.

In yet another preferred embodiment, steps (a) and (c) are performed ina temperature range from 20° C. to 37° C.

In a still further preferred embodiment, steps (a) and (c) are performedin a time range from 30 min to 120 min.

In another preferred embodiment of the method of the present invention,steps (a) and (c) are performed in a pH range from 7.0 to 9.0.

In a further preferred embodiment, the molar ratio of said first andsecond polypeptide is between 1:1 and 1:10.

In a more preferred embodiment of the method of the present invention,steps (a) and (c) are performed in a buffer comprising 0.15 M borate, pH8.0 at 37° C. for 30 min, the heterobifunctional crosslinker is used ina 30-fold higher molar concentration than said first polypeptide, andthe molar ratio of said first and second polypeptide is 1:10.

These conditions could be shown in accordance with the present inventionto result in conjugates that are most effective and beneficial if used,for example, to counteract allergic reactions.

In another preferred embodiment, the method of the present inventionfurther comprises the step of removing aggregated conjugate andproviding the conjugate in monomeric form.

Unexpectedly, this step even further improves the properties of theconjugates of the present invention.

In a still further preferred embodiment, said first polypeptide is animmunoglobulin or a structurally equivalent fragment thereof.

As discussed above, the method of the present invention may be, interalia, used to produce conjugates that, if comprising an allergen, may beused as a “tolerogen”, i.e. as a conjugate capable of inducingimmunologic tolerance in a subject allergic to the allergen itcomprises. Without wanting to be bound to a specific scientific theory,it is envisaged that these tolerogenic properties are conferred to theconjugate by a molecule that is also comprised by the conjugate, and isrecognized by the subject as “self” like, for example, an isologous orautologous immunoglobulin. Accordingly, the term “structurallyequivalent fragment” as used in accordance with the present inventiondenotes fragments that show the same immunological properties as theentire immunoglobulin, i.e. that do not induce an immune response in thesubject. Such a fragment may be, for instance, the Fc portion of animmunoglobulin.

In a more preferred embodiment, said immunoglobulin or structurallyequivalent fragment thereof is an immunoglobulin G or a structurallyequivalent fragment thereof.

In yet another preferred embodiment, said second polypeptide is anallergen, an autoantigen or an immunologically equivalent fragment ofsaid allergen or autoantigen.

“Immunologically equivalent fragment” as used in accordance with thepresent invention denotes a fragment of an allergen or autoantigen thatis capable of inducing the same immune response in a subject as thecorresponding allergen or autoantigen, i.e. the same allergic orautoantigenic reaction.

Since it could be shown that conjugates comprising an immunoglobulinand, for example, an allergen induce unresponsiveness in a subjectallergic to said allergen, it is envisaged in accordance with thepresent invention that conjugates comprising an immunoglobulin and, forexample, an autoantigen can be used to induce unresponsiveness to saidautoantigen and, thus, be used prophylactically in subjects suspected tohave a predisposition for the development of the correspondingautoimmune disease.

In a more preferred embodiment said allergen is derived from ragweed,birch pollen, peanut, house dust mite, animal danders, mould, or istropomyosin or an immunologically equivalent fragment thereof.

In another more preferred embodiment, said autoantigen is acetylcholinereceptor, insulin, insulin receptor, myelin basic protein or animmunologically equivalent fragment of these proteins.

In another embodiment, the present invention relates to a conjugateobtainable by the method of the present invention.

As could be shown in accordance with the present invention, theconjugates of the invention do not only protect subjects againsthypersensitivity type I reactions and, thus, the development of allergy.The conjugates of the inventions are also effective in downregulatingallergic reactions and, thus, may be used to treat allergic diseases.Likewise, it is envisaged that conjugates comprising an immunoglobulinand, for example, an autoantigen may be used to ameliorate and/or treatthe corresponding autoimmune disease and/or symptoms associatedtherewith.

The present invention also relates to a pharmaceutical compositioncomprising the conjugate of the present invention and, optionally, apharmaceutically acceptable carrier and/or diluent.

Examples of suitable pharmaceutical carriers are well known in the artand include saline solutions, water, emulsions, such as oil/wateremulsions, sterile solutions etc. Compositions comprising such carrierscan be formulated by well known conventional methods. Thesepharmaceutical compositions can be administered to the subject at asuitable dose. Administration of the suitable compositions may beeffected orally or parenterally, e.g., by intravenous, intraperitoneal,subcutaneous, intramuscular, topical, intradermal, intranasal orintrabronchial administration, or directly to the target site, e.g., bybiolistic delivery to an internal or external target site or by catheterto a site in an artery. The dosage regimen will be determined by theattending physician and clinical factors. As is well known in themedical arts, dosages for any one patient depends upon many factors,including the patient's size and weight, body surface area, age, theparticular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. A typical dose can be, for example, in the range of 0.001to 1000 μg. However, doses below or above this exemplary range areenvisioned, especially considering the aforementioned factors.Generally, the regimen as a regular administration of the pharmaceuticalcomposition should be in the range of 1 μg to 10 mg units per day. Ifthe regimen is a continuous infusion, it should also be in the range of1 μg to 10 mg units per kilogram of body weight per minute,respectively. Progress can be monitored by periodic assessment. Thecompositions of the invention may be administered locally orsystemically. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Furthermore, the present invention relates to the use of the conjugateof the present invention for the preparation of a pharmaceuticalcomposition for preventing and/or treating an allergic disease or anautoimmune disease.

In a preferred embodiment of the use of the present invention, whereinsaid allergic disease is an allergic reaction against an allergenderived from ragweed, birch pollen, peanut, house dust mite, animaldanders, mould, or tropomyosin or an immunologically equivalent fragmentthereof.

In another preferred embodiment, said autoimmune disease is Myastheniagravis, type I Diabetes or multiple sclerosis.

The Figures show:

FIG. 1: Primary challenge protocol. Mice were sensitized to Amba-1(ragweed extract) together with alum on days 1 and 14 and subsequentlychallenged with aerosolized (nebulized) Amba-1 on days 26, 27 and 28.Naïve (Nu) animals received no sensitization. IPNeb: intraperitonealsensitization/airway nebulization; Amba/AL: Amba-1/alum; AHR: airwayhyperresponsiveness; BAL: bronchoalveaolar lavage; PBLN: peribronchiallymph nodes; SC: spleen cells.

FIGS. 2a-b: Chromatography (top) and western blots (bottom) of theconjugates (a) conjugate 1 and (b) conjugate 2.

FIG. 3: Effect of conjugates on airway function. Penh: enhanced pause—ameasure of airway function.

FIGS. 4a-b: Effect of conjugates on eosinophil infiltration in thebronchoalveolar lavage fluid (BALF)(a) and serum antigen-specific IgElevels(b). EU/ml: Elisa units/ml.

FIG. 5: Effect of conjugates on BALF lymphocyte numbers(a), total IgE(b)and serum levels of antigen-specific IgG1(c) and IgG2(d).

FIG. 6: Effect of different conjugates on airway function.

FIG. 7: Secondary challenge protocol.

FIG. 8: Effect of different conjugates on airway function aftersecondary challenge.

The Examples illustrate the invention.

EXAMPLE 1

Preparation of a mouse IqG/Amba-l conjugate with either C₂₂H₃₁N₃O₁₁ orsulfo-GMBS

50 mg of mouse IgG (purchased from Prof. Ptak, Gracow Poland) weredissolved in 5 ml of 0.15 M borate buffer, pH 8.0. 100 μl of C₂₂H₃₁N₃O₁₁crosslinker were added at a concentration of 97 mM in ethanol, and thesample was mixed gently for 1 hr at room temperature. When sulfo-GMBSwas used instead of C₂₂H₃₁N₃O₁₁, 150 μl of sulfo-GMBS at a concentrationof 9 mM in H₂O were added to 50 mg of mouse IgG in 5 ml 0.15 M boratebuffer, pH 8.0 for 30 min at 37° C. Immediately thereafter excesscrosslinker was removed by gel filtration using a G25 Sephadex column in0.15 M borate buffer pH 7.5 with 10 mM EDTA. The fractions containingthe protein peak were collected and reduced to a volume of 3 ml.

At this point, the maleimide-activated protein may be used immediatelyin a conjugation reaction with a sulfhydryl-containing protein. If freeSH groups are not available, SATA reagent can be added on the allergen.

To do so, 4.8 mg of Amba-l (purified ragweed purchased from Greerlaboratory) were dissolved in borate buffer. 50 μl of SATA, 65 mM inDMSO were added, and the reaction mixture was incubated at roomtemperature for 30 minutes. Excess SATA was removed by gel filtration ona Sephadex G25 column in 0.15 M borate buffer pH 7.5 with 10 mM EDTA.Fractions containing Amba-l modified with SATA were collected andconcentrated in 6 ml. 600 μl of hydroxylamine, 0.5 M in borate buffer,10 mM EDTA were added to 6 ml Amba-l/SATA, and incubated for 2 hr atroom temperature with constant mixing. Excess hydroxylamine was removedon a G25 Sephadex column in 0.15 M borate buffer, 10 mM EDTA, pH 7.5.Fractions containing Amba-l modified with SATA and hydroxylamine werecollected and concentrated in 5 ml.

Mouse IgG (3 ml) and modified Amba-l (5 ml) were mixed and incubated for30 minutes at 37° C. with constant mixing. The mouse IgG/Amba-lconjugate was filtered before chromatography on a Sephacryl-S 300 HR in0.15 M borate buffer, NaN₃ 0.05%, pH 8.0, degassed under vacuum at 4° C.The fractions containing the conjugate were pooled and, after dialysisagainst 0.15 M NaCl, were concentrated in 4 ml (about 4 mg/ml).

If activation of the allergen with SATA is omitted, mouseIgG/C₂₂H₃₁N₃O₁₁ is mixed directly with 5 mg of Amba-l for 2 hr at roomtemperature and then for 30 minutes at 37° C. with constant mixing. Thenthe conjugate is filtered and chromatographed on Sephacryl-S 300 HR asdescribed above.

In each case, successful conjugation was verified by 8% polyacrylamidegel electroporesis and immunoblotting with specific antibodies (i.e.anti-IgG and anti-ragweed).

EXAMPLE 2

Sensitization and challenge

On days 1 and 14, Balb/c mice received intraperitoneal injections of 20μg Amba-l peptide (Greer Laboratories) together with 2 mg aluminiumhydroxide (Pierce). On day 26, 27 and 28 mice were exposed to a 20minutes aerosolization with a 0.2% solution of Amba-l in a closedplastic box. Airway responsiveness was assessed 48 hours after the lastchallenge by barometric plethysmography using whole body plethysmography(WBP) (Buxco, Troy, N.Y.) in live, unrestrained and non-ventilated micein response to inhaled methacholine (Mch) in a dose-response manner.Before taking readings, the box was calibrated with a rapid injection of150 μl air into the main chamber. Measured were pressure differencesbetween the main chamber of the WBP, containing the animal, and areference chamber. A pneumotachograph with defined resistance in thewall of the main chamber allows the measurement of flow (boxflow,pseudoflow) in and out of the main chamber. The measured box flowcorrelates in normal animals with the animal's flow and permits innon-bronchial constricted conditions the determination of tidal volumesand flow amplitudes (pseudovolume, pseudoflow) from box pressuresignals. In contrast to the original closed Fenn box, WBP uses apneumotachograph in the wall. Thus, the box pressure signal reflects boxflow changes due to respiration rather than box volume changes due torespiration, but still allows for determination of tidal volumes usingthe Fenn formula.

Inspiration and expiration are recorded by establishingstart-inspiration and end-inspiration as the box pressure curve crossesthe zero point. Start of an inspiration is determined by extrapolatingfrom a straight line drawn from two levels the rising inspiratory boxflow. In a pilot experiment, a non-bronchial constricted, consciousmouse was placed within a head-out body plethysmograph sealed with alatex collar and then placed into the current WBP. Comparison of thethoracic flow signal with the WBP flow signal showed a nearly identicalwave form with a slight (<10 ms) delay of start-inspiration of the WBPsignal and congruency of end-inspiration (personal communication, M.Lomask, Buxco). Time of inspiration (Ti) is defined as the time from thestart of inspiration to the end of inspiration; time of expiration (Te)as the time from the end of inspiration to the start of the nextinspiration. The maximum box flow occurring during one breath in anegative or positive direction is defined as peak inspiratory flow (PIF)or peak expiratory flow (PEF), respectively. Recordings of every tenbreaths are extrapolated to define the respiratory rate in breaths permin. The relaxation time (Tr) is defined as the time of volume decay ofthe pseudo-expiratory volume (area under the box flow waveform inexpiration) to 36%. This may thus serve as a correlate to the timeconstant (RC) of the decay of the volume signal to 36% of the peakvolume in passive expiration. During bronchoconstriction, the mainalteration occurs during early expiration and leads to changes in thewaveform of the box pressure signal. This change in the waveform can bequantified comparing the mean expiratory box flow during earlyexpiration (MF1) with the mean expiratory box flow during lateexpiration (MF2) by measurement of Pause, with:

MF1=mean pseudoflow 1

MF2=mean pseudoflow 2

V=pseudo-expiratory volume

MF1=0.65 V/Tr

MF2=0.35 V Te−Tr

Pause=Te−Tr/Tr−0.35 V/0.65 V×MF1/MF2−MF1/MF2

During bronchoconstriction, the changes in box flow during expiration(PEF) are more pronounced than during inspiration (PIF). This isreflected by the formula for enhanced Pause (Penh), a dimensionlessvalue used in this paper to empirically monitor airway function:

Penh=Pause×PEF/PIF

Penh reflects changes in the box flow waveform from both inspiration andexpiration (PIF, PEF) and combines it with the comparison of early andlate expiratory box flow (Pause). Penh is not a function of the absolutebox flow amplitude or the respiratory rate, but rather of the proportionof inspiratory to expiratory flow and of the timing of expiration.

Mice were placed in the main chamber and baseline readings were takenand averaged for three minutes. Aerosolized PBS or methacholine inincreasing concentrations (3 to 50 mg/ml) were nebulized through aninlet of the main chamber for three minutes and readings were taken andaveraged for three min following each nebulization. Airway reactivitywas expressed as a fold increase for each concentration of MCh(Penh_(MCh)) compared to Penh values after PBS challenge (Penh_(PBS)).

For the quantification of the dose-response to methacholine, the linearregression of Penh on log base 2 was calculated for individual mice. Thelog dose corresponding to an increase in Penh of 100% or 200%,respectively, was determined and the average log doses of the differentgroups were compared by analysis of variance. The data are reported asthe geometrical mean with the lower and upper limit of the 95%confidence interval.

Conjugates were given intravenously on days 1, 7 and 14 at a dose of 500μg.

Serum was obtained 48 hours after the last challenge (after airwayfunction is assessed) and total IgE and antigen specific IgE, IgG1, IgG2were measured by ELISA. Bronchoalveolar lavage fluid (BALF) wascollected after measurements of airway function and eosinophil numbersas well as total lymphocyte counts were determined. A summary of atypical experimental protocol is presented in FIG. 1. Each groupconsisted of 5 to 6 animals.

EXAMPLE 3

Bronchoalveolar lavage (BAL), lung cell isolation

Lungs were lavaged via a tracheal tube with Hank's balanced saltsolution (HBSS, 3×0.5 ml) and the cells in the lavage fluid werecounted. Cells from BAL or lungs were resuspended in HBSS and countedwith a hemocytometer. Cytospin slides were stained with Leukostat(Fisher Diagnostics) and differentiated in a blinded fashion by countingat least 300 cells by light microscopy.

EXAMPLE 4

Measurement of anti-OVA antibody and total Ig levels

Anti-Amba-1 Ig serum levels were measured by ELISA. The antibody titersof the samples were related to pooled standards that were generated inthe laboratory and expressed as ELISA units per ml (EU/ml). Total IgEand IgG levels were determined using the same method compared with knownmouse IgE or IgG standards (PharMingen, San Diego, Calif.). The limitsof detection were 100 pg/ml for IgE and 1 ng/ml for IgG.

EXAMPLE 5

Comparison of C₂₂H₃₁N₃O₁₁ conjugates with sulfo-GMBS conjugates

Profile 1 and Profile 2 of FIG. 2 represent the elution profilesobtained by chromatography of the C₂₂H₃₁N₃O₁₁ conjugate (conjugate 1)and the sulfo-GMBS conjugate (conjugate 2), respectively. In both casesthe conjugates react with either anti-IgG or anti-ragweed demonstratingthe covalent binding of Amba-l to mouse IgG as seen on immunoblots.

Administration of conjugate 1 and 2 has a markedly different influenceon airway function. Conjugate 1 leads to a significant reduction ofairway function whereas conjugate 2 has almost no effect when comparedto sensitized animals (positive control) (see FIG. 3). Likewise,conjugate 1 virtually eliminated eosinophils in BALF. This wasaccompanied by a total suppression of specific anti Amba-l IgE (FIG. 4).As expected, total IgE was unaffected by treatment as well as totallymphocyte count. There was a difference as far as specific IgG1 andIgG2 is concerned. Administration of either conjugate 1 or 2 resulted inan increase of IgG1 anti-Amba-l antibodies. In contrast, conjugate 1appears to reduce anti IgG2 antibodies as in comparison to conjugate 2(FIG. 5).

The increase in the IgG1 specific isotype in contrast to the decrease inIgG2 following administration of the conjugate is interesting. It couldbe due to several factors: (a) the dose of the conjugate, the animalspecies, a difference in susceptibility of the various immune responsesfollowing administration of tolerogen. A phenomenon reminiscent of whathas been referred to as “immune deviation” or “split tolerance”; Borelet al., J. Exp. Med 131 (1970), 603; (b) whether suppression of allimmune responses as shown for both cellular and humoral immunity; Borelet al., Parenteral and oral administration of Tolerogens i.e. ProteinIgG conjugates, Proc. N.Y. Acad. of Sci. 778 (1996), 80-87; or whetherthe formation of IgG1 specific “blocking” antibody would be desirable todownregulate allergic reactions remains to be determined.

EXAMPLE 6

Influence of the amount of Amba-l used for crosslinking on theeffectiveness of the conjugate

The results in FIG. 6 show that 5 mg Amba-l was effective as far as thediminution or normalization of airway hyperresponsiveness is concerned(conjugate 3). In contrast, 2.5 mg was not (conjugate 4). Interestingly,for the same dose of Amba-l (5 mg) there was essentially no differencewhether or not SATA was used before crosslinking (FIG. 6).

EXAMPLE 7

Effect of the conjugate on previously sensitized allergic animals

It was determined whether the conjugate is effective not only in naivebut also in previously sensitized allergic animals. The protocolfollowed is shown in FIG. 7. The results with conjugates 6 and 7 clearlydemonstrate that these conjugates downregulate sensitized mice.

EXAMPLE 8

Effect of the type of immunoglobulin used for conjugate synthesis

Monoclonal antibody with two different isotypes γ₁ and γ_(2a) as thecarrier molecule was tested, as compared to polyclonal IgG. The resultsdemonstrate that while polyclonal IgG (conjugate 8) shows partialsuppression, γ₁ (conjugate 9) was ineffective. In contrast, γ_(2a)(conjugate 10) downregulates airway responsiveness in atopic asthma.

We claim:
 1. A method for the preparation of a conjugate comprising animmunoglobulin or a structurally equivalent fragment thereof and anallergen or autoantigen, said method comprising the steps of: (a)incubating said immunoglobulin or said structurally equivalent fragmentthereof in the presence of a heterobifunctional crosslinker comprisingan N-hydroxylsuccinimide ester group and a maleimide group linked via apolyethylene oxide spacer; (b) removing excess heterobifunctionalcrosslinker; and (c) incubating the reaction product of step (b) withsaid allergen or autoantigen, wherein said allergen or autoantigencomprises at least one sulfhydryl group.
 2. The method of claim 1,wherein said at least one sulfhydryl group of said allergen orautoantigen is introduced by: (i) incubating said allergen orautoantigen in the presence of N-succinimidyl-S-acetylthioacetate(SATA); (ii) removing excess SATA; (iii) incubating the reaction productof step (ii) in the presence of hydroxylamine; and (iv) removing excesshydroxylamine and acetylated hydroxylamine.
 3. The method of claim 1,wherein said heterobifunctional crosslinker comprising anN-hydroxylsuccinimide ester group and a maleimide group linked via apolyethylene oxide spacer has the formula:


4. The method of claim 1, wherein in step (a) the heterobifunctionalcrosslinker is used in a 5- to 50-fold higher molar concentration thansaid immunoglobulin or said structurally equivalent fragment thereof. 5.The method of claim 1, wherein steps (a) and (c) are performed in atemperature range from 20° C. to 37° C.
 6. The method of claim 1,wherein steps (a) and (c) are performed in a time range from 30 min to120 min.
 7. The method of claim 1, wherein steps (a) and (c) areperformed in a pH range from 7.0 to 9.0.
 8. The method of claim 1,wherein the molar ratio of said immunoglobulin or said structurallyequivalent fragment thereof compared to said allergen or autoantigen isbetween 1:1 and 1:10.
 9. The method of claim 4, wherein steps (a) and(c) are performed in a buffer comprising 0.15 M borate, pH 8.0 at 37° C.for 30 min, the heterobifunctional crosslinker is used in a 30-foldhigher molar concentration than said immunoglobulin or said structurallyequivalent fragment thereof, and the molar ratio of said immunoglobulinor said structurally equivalent fragment thereof compared to saidallergen or autoantigen is 1:10.
 10. The method of claim 1 furthercomprising the step of removing aggregated conjugate and providing theconjugate in monomeric form.
 11. The method of claim 1, wherein saidimmunoglobulin or said structurally equivalent fragment thereof is animmunoglobulin G or a structurally equivalent fragment thereof.
 12. Themethod of claim 1, wherein said allergen is derived from ragweed, birchpollen, peanut, house- dust mite, animal danders, or mould, or istropomyosin.
 13. The method of claim 1, wherein said autoantigen isacetylcholine receptor, insulin, insulin receptor, myelin basic protein.